Method for treatment of human eye disease

ABSTRACT

Sterile aqueous humor from patients afflicted with the disease endogenous uveitis contains a previously unreported pathogenic microorganism, Micromyces intracellularis, which also has been found in tissue specimens from other diseases. When inoculated into an animal model (e.g., a mouse), the microorganism produces both chronic eye and systemic disease in addition to accelerated mortality and occasional psychotic animal behavior, metabolic disturbances, and neuro-muscular disorders, depending on the mode of inoculation. The animal model therefore provides the basis for a screening process to evaluate chemotherapeutic agents in the treatment of human disease. Using this animal model, a significant decrease in mortality associated with the introduction of Micromyces intracellularis is achieved by the application to the animal model of the chemotherapeutic agents d-2,2&#39;-(ethylenediimino)-di-1-butanol, 4,4&#39;sulfonyldianiline, and 3[[(4-methyl-1-piperazinyl)-imino]-methyl]-rifamycin SV. These agents are therefore effective in the treatment of diseases having as their etiological agent Micromyces intracellularis, and are particularly useful in the chemotherapy of endogenous uveitis, conjunctivitis and kerato-conjunctivitis.

This is a division of application Ser. No. 932,904, filed Aug. 11, 1978,now U.S. Pat. No. 4,220,657, which is a division of application Ser. No.453,463, filed Jan. 23, 1974, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the treatment of human disease. Moreparticularly, it relates to the recovery and identification of ahitherto unknown infectious microorganism, the application of thispathogen to laboratory animals, and to novel indications for the use ofcertain known chemotherapeutic agents in the treatment of human diseasesassociated with the novel pathogen.

The search for new therapeutic agents and novel indications for existingdrugs in the treatment of human diseases requires an animal model systemrelated to each particular disease under investigation and which isresponsive to a reproducible source of the infectious agent. Non-treatedinfected animals of the model must show symptoms or succumb to thedisease in a regular, predictable manner. In addition, although somelaboratory animals inoculated with a human disease-causing agent mayshow a syndrome with a long latent period, it must be possible todefinitely associate the syndrome with the corresponding human chronicdisease.

The isolation of a causative agent from human chronic inflammatorydisease conditions capable of inducing those conditions in an animalmodel would therefore be an important breakthrough in the etiology ofsuch human diseases.

Accordingly, it is an object of the present invention to provide aprocess for recovering an infectious microorganism from inflammatorydisease tissue or fluid as the causative agent capable of inducing thosedisease conditions.

Another object of the present invention is to provide an animal modelsystem responsive in a definite and predictable manner to a humandisease-causing agent.

Another object is to provide novel indications for existing drugs andmeans for designing new therapeutic agents by means of an animal model.

These and other objects of the present invention as well as a fullerunderstanding of the advantages thereof can be had by reference to thefollowing detailed description and claims.

DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that sterile aqueoushumor (eye fluid) from patients afflicted with endogeneous uveitiscontains a unique and heretofore unreported pathogenic microorganismwhich can be observed with the light microscope. This pathogen has acomplex life cycle, is not propagatable by the usual in vitro laboratorytechniques, and when inoculated, say, subconjunctivally into one or botheyes of a host animal ("animal model"), produces both eye and systemicdiseases, the latter being generally randomly distributed inflammatorydiseases of any organ system and less commonly either degenerative orneoplastic in type. The accelerated mortality induced by the pathogen isaccompanied occasionally by psychotic animal behavior, metabolicdisturbances with glucosuria, and neuro-muscular disorders characterizedby coarse head tremors, paresis, motor seizures and/or locomotor ataxia.

The new microorganism, which we have designated Micromycesintracellularis, has been recovered and identified from the inflammedaqueous humor of patients suffering from endogeneous uveitis. It differsfrom any of the known phyta listed in the accepted classification ofBergey's Manual of Determinative Bacteriology (7th edition, 1957).Individual microorganisms in this taxonomic system are distinguished,for purposes of classification, on the basis of morphologicalcharacteristics and biochemical behavior. Accordingly, it is believedthat this novel organism constitutes a new family, Micromycetaceae,within the Order Actinomycetales because it has properties in commonwith other species of this order. A living culture of the organism hasbeen deposited with the American Type Culture Collection at Rockville,Maryland and has been added to its permanent collection ofmicroorganisms as ATCC 31,000.

The organism or agent cannot be propagated in vitro on bacteriologicalmedia, whether enriched or selective. A large variety of media have beentested, using varying atomospheric and temperature conditions, with nodemonstrable growth after prolonged incubation, even up to twelvemonths. A large variety of tissue cultures also fail to propagate theorganism.

The organism has a complex growth cycle, both intra- andextracellularly, during which a wide variety of morphologicallydistinctive parts are produced. These parts have quite variabletinctorial properties.

In a fresh aqueous suspension of Micromyces intracellularis small coccalparticles can be observed which are approximately 1 to 2 microns indiameter, and have a distinctive, small amplitude, rapid, back-and-forthmotility, which differs from Brownian movement. This motility is readilyappreciated with both dark field and phase contrast microscopictechniques.

The use of tissue smears is the best way to demonstrate the completelife cycle of the agent. The organism parasitizes a wide variety of bothfixed and wandering cells. This is best appreciated in epithelial andendothelial cells where a large number of varying sized, inclusion-likeparticles can be seen within the cytoplasm in the earliest intracellulargrowth phase of the organism. One parasitized cell may contain many suchparticles. The smallest of these particles are often Gram positive,generally acid or basis staining, and Periodic Acid Schiff positive.These particles continue to develop intracellularly. Some appeardestined to become larger intracytoplasmic coccal forms, whereas othersdevelop into very fine filaments, a fraction of a micron in diameter,and into mats of these filaments. These fine filaments grow in lengthwithout any visible internal structure and with variable stainingproperties. Extracellularly, a filament may extend beyond theparasitized cell boundary for great distances where it undergoes furtherdevelopment and differentiation with the production of bacterial-shapedforms. As the filaments develop extracellularly they may showdistinctive internal structure with vacuolization and bands being seen.The extracellular filaments also give rise to bacilli. The smallestbacilli may resemble safety pins in appearance. The largest bacilliproduced are long, thick, vacuolated tubes. These bacillary bodies andtubes are generally Gram negative and have an affinity for acidophilicstains. Some of the most delicate filaments may be partly Periodic AcidSchiff positive and weakly acid fast. Coccal particles arising from theextracellular filament are quite distinctive morphologically. These canarise from the filament, like a row of peas, at varying intervals. Thesmallest of these coccal bodies is less than one micron in diameter, andthe largest may be the size, say, of a polymorphonuclear leukocytenucleus. Many of these extracellularly developing cocci appear toenlarge and to divide into pairs and tetrads with biscuit shapes. Thesecocci are generally Gram negative and only rarely Gram positive. Theseextracellularly developing cocci stain variably with both acid and basicstains, and they may be Periodic Acid Schiff positive. Although thesmallest cocci are homogeneous in their staining, the larger cocci haveinternal structure demonstrable with the usual stains. At the terminusof the delicate, undifferentiated, extracellular portion of some of thefilaments a cluster of spherical, uniform sized, tiny particles, similarto those seen in the cytoplasm of the parasitized cell, are occasionallyseen.

In fixed tissue sections the finer intra-cytoplasmic forms describedabove are difficult to demonstrate. The filaments are most easilyrecognized when a large extra-cellular cluster of these stain basic withthe usual tissue stains, such as Hematoxylin, giving the appearance of adense mat. The pleomorphic cocci described above are widely distributedin varying numbers against this compact filamentous background. Thelarger cocci, because of their varying large size and basophilicstaining properties, are indistinguishable from nuclear debris. Largeclusters of the smaller, uniform-staining cocci resemble atypicaleosinophil or basophil granules. The larger bacilli and sausage-shapedfilaments are often difficult to recognize because they fail to stainwell. When they do stain well with the basophilic stains, they mayresemble compressed connective tissue nuclei. The Periodic Acid Schiffstain or silver stain, such as Gomori's Mathenamine Silver Stain, has avarying affinity for different internal structures, particularly in thelarger cocci and filamentous structures, producing a granular orreticulated appearance. The outline of the individual forms aredifficult to appreciate as they arise from the site of the parasitizedgroups of cells.

A wide variety of clinical (sterile) specimens containing the agentMicromyces intracellularis retain their infectivity after passingthrough millipore filters (e.g., Seitz-type filters), which normallyprevent the passage therethrough of bacterial-size particles. Examplesof such specimens include aqueous emulsions of colon carcinomas,hypertensive kidneys, arteriosclerotic aortic atheromas, myocardialinfarctions, inflamed synovia from rheumatoid arthritis joints, lenscataracts, and body fluids such as diabetic urine and spinal fluid frompatients with demyelinating diseases and psychiatric disorders. Filteredclinical specimens also retain their infectivity following storage in acell-free, aqueous state at ordinary refrigerator temperatures (e.g., atbetween 4° and 7° C.) for up to twelve months and perhaps longer.Infectivity of the organism can be destroyed by autoclaving (15p.s.i.g./15 minutes/212° F.), by boiling for 10 minutes, or by treatmentwith phenol.

The pathogen described hereinabove has been found in acute and chronicendogeneous uveitis, acute and chronic non-specifickeratoconjunctivitis, chronic keratitis, chronic conjunctivitis, chroniccorneal ulcer, orbital cellulitis and may be found in non-specificinflammtory diseases of any human organ system.

Host organisms which can serve as the animal model of the presentinvention include, without limitation, fowl (e.g., chickens), primates(e.g., chimpanzees), rodents (e.g., mice, rats, rabbits, guinea pigs),cattle, horses, pigs, dogs, cats and the like.

Inoculation of Micromyces intracellularis into sites of the animal modelother than the eye produces local inflammatory and, less frequently,degenerative or neoplastic lesions, in addition to the ocular andsystemic diseases and accelerated mortality described earlier and ingreater detail hereinbelow. By "inoculation," it is understood to meanthe term as it is commonly used in the medical arts, including oralinoculation, parenteral inoculation (e.g., intravenous, intra-articular,subcutaneous, intra-peritoneal, intradermal, intramuscular andintrathecal inoculation) and topical inoculation (e.g., mucosal,conjunctival, and dermal application). The modes of inoculationdescribed above are also applicable to the administration ofchemotherapeutic agents in the treatment of diseases having as theiretiological agent the newly-discovered microorganism describedhereinabove.

In measuring the response of the animal model inoculated with Micromycesintracellularis, any of a number of recognized methods can be used,including mortality, general morbidity, visual inspection of morbidityat the site of inoculation, histology, and quantitation of theinfectious agent present in the animal model tissue.

INOCULATION OF THE ANIMAL MODEL

The following procedures illustrate the techniques for inoculating micewith sterile materials containing Micromyces intracellularis as ananimal model according to the present invention. It should be understoodthat the procedures described hereinbelow can be varied within the scopeof the present invention and are generally applicable to any hostorganism suitable for use in the animal model of the present invention.

I. PATHOGENICITY OF ORGANISM INFECTED INFLAMED HUMAN AQUEOUS HUMOR

A. Sterile inflamed aqueous humor from 100 patients with acutelyrecurrent chronic endogenous uveitis was obtained by anterior chamberparacentesis. Each specimen was stored at 4° C. for 4 to 12 months, andindividually inoculated, 0.05 cc per eye, subconjunctivally into eacheye of 100 AKR 8 to 10 gram male mice.

B. Sterile aqueous humor from 100 eye bank eyes, observed to be grosslynormal, was obtained by sterile paracentesis. In some cases, thismaterial was composed of liquid-vitrous-aqueous mixture. Each specimenwas stored at 4° C. for 4 to 12 months, and individually inoculated,0.05 cc per eye, subconjunctivally into each eye of 100 AKR 8-10 grammale mice.

C. Sterile isotonic saline, 0.05 cc per eye, was subjunctivallyinoculated into each eye of 100 AKR 8 to 10 gram male mice.

II. PATHOGENICITY OF ORGANISM INFECTED DIABETIC RETINOPATHY URINE

A. Sterile urine from 10 patients with Diabetic Retinopathy was storedat 4° C. for 4 to 12 months, and individually inoculated, 0.05 cc pereye, subconjunctivally into each eye of 100 AKR 8 to 10 gram male mice.

B. Sterile urine from 10 normal young volunteers was stored at 4° C. for4 to 12 months, and individually inoculated, 0.05 cc per eye,subconjunctivally into each eye of 100 AKR 8 to 10 gram male mice.

C. Sterile isotonic saline, 0.05 cc per eye, was inoculatedsubconjunctivally into each eye of 100 AKR 8 to 10 gram male mice.

III. PATHOGENICITY OF ORGANISM INFECTED MULTIPLE SCLEROSISCEREBRO-SPINAL FLUID

A. Sterile inflamed cerebro-spinal fluid from 10 institutionalizedpatients with active multiple sclerosis was stored at 4° C. for 4 to 12months, and individually inoculated, 0.05 cc per eye, subconjunctivallyinto each of 100 AKR 8 to 10 gram male mice.

B. Sterile isotonic saline, 0.05 cc per eye, was subconjunctivallyinoculated into each eye of 100 AKR 8-10 gram male mice.

IV. PATHOGENICITY OF ORGANISM INFECTED CATARACTOUS LENS HOMOGENATE

A. Mature cataracts from 10 patients, each emulsified in steriledistilled water (1:10) was stored at 4° C. for 4-12 months, tested forfinal bacterial sterility, and individually inoculated, 0.05 cc per eye,subconjunctivally into each eye of 100 PARIS 8-10 gram male mice.

B. Normal clear lenses from 10 eye bank eyes, each emulsified in steriledistilled water (1:10), was stored at 4° C. for 4-12 months, tested forfinal bacterial sterility, and individually inoculated, 0.05 cc per eye,subconjunctivally into each eye of 100 PARIS 8-10 gram male mice.

C. Sterile isotomic saline, 0.05 cc per eye, was subconjunctivallyinoculated into each eye of 100 PARIS 8-10 gram male mice.

V. PATHOGENICITY OF ORGANISM INFECTED CHRONIC HUMAN CORNEAL STROMALULCERS

A. Inflamed corneal stroma was obtained by keratectomy from 10 patientswith chronic sterile corneal stromal ulcers. Each specimen wasemulsified in sterile distilled water (1:10), filtered (using aMillipore Corp. SXGS 025 0.22 μm filter), stored at 4° C. for 4-12months, tested for final bacterial sterility, and individuallyinoculated, 0.05 cc per eye, subconjunctivally into each eye of 100PARIS 8-10 gram male mice.

B. Sterile corneal stroma was obtained from 10 normal appearing cadavercorneas. Each specimen was emulsified in sterile distilled water (1:10),filtered as in V(A), above, stored at 4° C. for 4-12 months, tested forfinal bacterial sterility, and individually inoculated, 0.05 cc per eye,subconjunctivally into each eye of 100 PARIS 8 to 10 gram male mice.

C. Sterile isotonic saline, 0.05 cc per eye, was subconjunctivallyinoculated into each eye of 100 PARIS 8 to 10 gram male mice.

VI. PATHOGENICITY OF ORGANISM INFECTED RHEUMATOID ARTHRITIS JOINTS

A. Synovial biopsy material was taken from ten patients with ActiveRheumatoid Arthritis. Each inflamed specimen was emulsified in steriledistilled water (1:10), millipore filtered, stored at 4° C. for 4 to 12months, tested for final bacterial sterility, and individuallyinoculated, 0.05 cc per eye, subconjunctivally into each eye of 100 AKR8 to 10 gram male mice, 10 mice per specimen.

B. Synovial tissue was obtained from ten cadaver knee joints with nogross and histological evidence of joint disease. Each specimen wasemulsified in sterile distilled water (1:10), filtered as above, storedat 4° C. for 4 to 12 months, tested for final bacterial sterility, andindividually inoculated, 0.05 cc per eye, subconjunctivally into eacheye of 100 AKR 8 to 10 gram male mice, 10 mice per specimen.

C. Sterile isotonic saline, 0.05 cc per eye, was subconjunctivallyinoculated into each eye of 100 AKR 8 to 10 gram male mice.

VII. PATHOGENICITY OF ORGANISM INFECTED COLON CARCINOMAS

A. Colon carcinoma tissue was obtained from 10 patients with surgicallyresected specimens. Approximately 3 to 4 grams of the fungating and/orulcerated portion of the tumor was emulsified in sterile distilled water(1:10), filtered as above, stored at 4° C. for 4 to 12 months, testedfor final bacterial sterility, and individually inoculated, 0.05 cc pereye, subconjunctivally into each eye of 100 AKR 8-10 gram male mice, 10mice per specimen.

B. Normal colonic mucosa was obtained from 10 grossly and histologicallynormal cadaver colons. Approximately 3-4 grams of the normal colon wallwas emulsified in sterile distilled water (1:10), filtered as above,stored at 4° C. for 4-12 months, tested for final bacterial sterility,and individually inoculated, 0.05 cc per eye, subconjunctivally intoeach eye of 100 AKR 8-10 gram mice, 10 mice per specimen.

C. Sterile isotonic saline, 0.05 cc per eye, was subconjunctivallyinoculated into each eye of 100 AKR 8-10 gram male mice.

VIII. PATHOGENICITY OF ORGANISM INFECTED STERILE POOLED GUINEA PIGPASSAGE EYE EMULSIONS

A. Guinea pig eyes from 100 guinea pigs, which died 3-6 months aftersubconjunctival inoculation with 0.1 cc of inflamed aqueous humor from10 individual patients with Acute Exacerbations of Chronic EndogenousUveitis, were pooled and emulsified (1:10) in sterile distilled water,filtered as above, stored at 4° C. for 4-12 months, tested for finalbacterial sterility, and subconjunctivally inoculated, 0.05 cc per eye,into each eye of 100 AKR 8-10 gram male mice. (The presence ofMicromyces intracellularis was verified in each of the 10 pairs ofguinea pig eyes).

B. An autoclaved aliquot of the ten Group VIII-A emulsified guinea pigeyes was filtered as above, stored at 4° C. for 4-12 months, tested forfinal bacterial sterility, and subconjunctivally inoculated, 0.05 cc pereye, into each eye of 100 AKR 8-10 gram male mice.

C. A phenolized aliquot of the ten Group VIII-A emulsified guinea pigeyes (0.5 cc of phenol per 25 cc of aqueous emulsion incubated for 18hours) was filtered as above, stored at 4° C. for 4-12 months, testedfor final bacterial sterility, and subconjunctivally inoculated, 0.05 ccper eye, into each eye of 100 AKR 8-10 gram male mice.

D. An aliquot of the ten Group VIII-A emulsified guinea pig eyes wasboiled for 10 minutes, filtered as above, stored at 4° C. for 4-12months, tested for final bacterial sterility, and inoculated, 0.05 ccper eye, into each subconjunctiva of 100 AKR 8-10 gram male mice.

E. A pair of eyes from each of 10 guinea pigs, which were sacrificed 6months after individual subconjunctival inoculation of 0.1 cc of aqueoushumor from 10 grossly normal eye bank eyes, was individually emulsified(1:10) in sterile distilled water, filtered as above, stored at 4° C.for 4-12 months, tested for final bacterial sterility, and individuallyinoculated subconjunctivally, 0.05 cc per eye, into each eye of 10 AKR8-10 gram male mice. (The presence of Micromyces intracellularis couldnot be detected in any of the ten specimens.)

F. Sterile isotonic saline, 0.05 cc per eye, was inoculatedsubconjunctivally into each eye of 100 AKR 8-10 gram male mice.

The observed pathogenicities resulting from the abovedescribedinoculations are summarized in Table I, below.

                                      TABLE I                                     __________________________________________________________________________    Pathogenicities Observed in the Mouse Animal Model                            PERCENT         STATISTICAL SIGNIFICANCE                                      MORTALITY AT    OF OBSERVED DIFFERENCES                                       GROUPS                                                                              12 MONTHS GROUPS       CHI-SQUARE*                                      __________________________________________________________________________    I(A)  95%       I(A) vs. I(B)                                                                              92.9                                             I(B)  30%       I(A) vs. I(C)                                                                              108                                              I(C)  25%       I(B) vs. I(C)                                                                              0.9                                              II(A) 82%       II(A) vs. II(B)                                                                            53.05                                            II(B) 32%       II(A) vs. II(C)                                                                            67.6                                             II(C) 25%       II(B) vs. II(C)                                                                            1.5                                              III(A)                                                                              65%       III(A) vs. III(B)                                                                          33.95                                            III(B)                                                                              25%         --         --                                               IV(A) 66%       IV(A) vs. IV(B)                                                                            14.6                                             IV(B) 40%       IV(A) vs. IV(C)                                                                            21.73                                            IV(C) 34%       IV(B) vs. IV(C)                                                                            0.53                                             V(A)  78%       V(A) vs. V(B)                                                                              39.2                                             V(B)  33%       V(A) vs. V(C)                                                                              52.1                                             V(C)  26%       V(B) vs. V(C)                                                                              0.865                                            VI(A) 62%       VI(A) vs. VI(B)                                                                            23.3                                             VI(B) 29%       VI(A) vs. VI(C)                                                                            29.38                                            VI(C) 25%       VI(B) vs. VI(C)                                                                            3.06                                             VII(A)                                                                              64%       VII(A) vs. VII(B)                                                                          24.66                                            VII(B)                                                                              28%       VII(A) vs. VII(C)                                                                          32.39                                            VII(C)                                                                              25%       VII(B) vs. VII(C)                                                                          0.41                                             VIII(A)                                                                             96%       VIII(A) vs. VIII(B)                                                                        93.85                                            VIII(B)                                                                             22%       VIII(A) vs. VIII(F)                                                                        113.6                                            VII(C)                                                                              25%       VIII(B,C,D) vs. VIII(F)                                                                    0.111                                            VIII(D)                                                                             24%         --         --                                               VIII(E)                                                                             31%       VII(E) vs. VIII(F)                                                                         2.05                                             VIII(F)                                                                             23%         --         --                                               __________________________________________________________________________     ##STR1##                                                                      where                                                                         fo = observed mortality rate                                                  f = expected (control mortality rate                                     

APPEARANCE OF THE ANIMAL MODEL AFTER INOCULATION WTH CLINICAL HUMANMATERIALS CONTAINING MICROMYCES INTRACELLULARIS I. DIRECT EYEINOCULATION

A. Mice

During the first two weeks following inoculation of pretreated clinicalmaterials containing Micromyces intracellularis subconjunctivally into8-10 gram mice, no change is noted in the inoculated eyes or in theanimal. During the 3rd or 4th week an occasional mouse (about 10%)demonstrates one or both eye lids closed by exudate for varying periodsof time, usually several days, and about 5% demonstrate abnormalsystemic changes such as decreased motor activity, dull coats, orbizarre motor activity. Increased mortality relative to control micebegins to be seen during the 3rd or 4th week following inoculation. Bythe end of the fourth week usually about 10% of the inoculated mice havedied. During the next four weeks more eyes become closed, either one orboth, for varying periods of time (usually several days but some eyesbecome permanently closed, so that by the end of the 8th week usuallyabout 30-50% of the inoculated mice are affected. At this time usuallyabout 20% of the inoculated mice will have died. This increasedmortality rate among inoculated mice versus the mortality rate amongcontrol mice continues at a constant rate over the succeeding 44 weeksso that by the end of 12 months usually 65-95% of the inoculated mice(the exact percentage presumably depending on the initial concentrationof the inoculum and variations in the host response) have died.Following inoculation, an occasional mouse will develop among otherthings, a pthisis bulbi, proptosis, a hematocele, or intra- andperiocular neoplasms. About 5% of the mice develop red, swollen jointsand about an equal number demonstrate glucosuria, gait or locomotorabnormalities, motor seizures, or course head tremors either transientlyor permanently. A graphical representation of the observed increase inmortality rate among the mice which were inoculated with Micromycesintracellularis according to procedures I(A)-(B), above, is presented inthe accompanying drawing. It can be seen from the "survival curves" inthe drawing that, by using an appropriate number of animals in the modelsystem of the present invention (i.e., controls and experimentals), therequired period of observation can, if desired, be appreciablyshortened.

B. Guinea Pigs

During the first month following subconjunctival inoculation of sterileclinical materials containing Micromyces intracellularis into 150 gramguinea pigs, no ocular or systemic lesions are noted, but during thesecond month about 5% of the guinea pigs develop ocular suppurationcharacterized by hyperemic conjunctiva, sticky eye lids in the morning,and purulent discharge. Occasionally this rapidly progresses to aPhthisis bulbi. During the second month mortality begins to be seen. Bythe end of the third month about 10% of the guinea pigs develop eyesuppuration and systemic lesions begin to be seen, such as weight loss,generalized lymphadenopathy, and hot, tender swollen joints. Mortalitybegins to increase during this time, and by the end of the fourth monthanother 10% of the inoculated animals show these systemic signs ofdisease. About 5% of the inoculated guinea pigs by the end of the fourthmonth demonstrate paresis of one or more extremities, a hemiparesisoccasionally being seen. An equal number at this time demonstratecoarse, rhythmic head tremors, and about 5% during the third to fourthmonth will demonstrate glucosuria and occasionally hematuria and/orproteinuria. Usually by the sixth month the mortality among inoculatedguinea pigs is 75-100%. (Symptoms of Conjunctivitis, Arthritis,Lymphadenitis, and wasting were not seen in control guinea pigs, wherethe mortality rate varied from 0-50% at six months).

II. Subcutaneous Inoculation at Sites Distant from the Eye

A. Mice

Subcutaneous inoculation of sterile clinical materials containingMicromyces intracellularis into the facial skin region results in puffy,red, swollen facial tissues in about 25% of the mice 2 to 4 weeksfollowing inoculation. Facial alopecia at the inoculation site isoccasionally seen during this time period. These lesions usuallydisappear by the sixth week but an occasional eye is seen to be closedfor short periods of time prior to this. Mortality during the ensuingtime period is similar to that described in the subconjunctivalinoculation route.

When sterile clinical materials containing Micromyces intracellularisare inoculated subcutaneously in the skin of the sacral region, thereresult multiple erythematous plaques, macules, papules, pustules, and/orulcers of the tails in about 25% of the mice 2-4 weeks afterinoculation. Healing of these lesions is slow with occasional nodular,deformed tails being seen several weeks later. Other affected tailsbecome gangrenous and slowly fall off, either at the tip or at otherpoints between the tip and the base of the tail. About 25% of the micedevelop, either with or without the above described tail disease,varying sized areas of alopecia, erythema, yellow greasy coats and/orulcers at the site of inoculation. These frequently heal slowly duringthe ensuing weeks, but in many of the affected mice these lesions recur.Some of the affected mice became so debilitated in appearance, thatdeath appears imminent during the 3rd and 4th month. However, some ofthese severely debilitated mice do survive as runts after 12 months ofobservation. About 10% of the mice so inoculated demonstrate one or botheyelids closed for several days at a time sometime during the course ofobservation. About 5% of the inoculated mice demonstrate red swollenjoints for varying and protracted periods at various times during thecourse of observation, and about a similar percentage demonstratebizarre motor activity, paresis, head tremors, or seizures, at one timeor another during the course of observation. The 12-month mortality inthe mice so inoculated is similar to that described for thesubconjunctivally inoculated mice.

B. Guinea Pigs

The subcutaneous inoculation of sterile clinical materials containingMicromyces intracellularis does not result in observable cutaneouslesions during six months of observation. Lymphadenopathy and increasedmortality such as described for the subconjunctival inoculation route isseen, however.

III. Intraperitoneal Inoculation

A. Mice

During the first week following intraperitoneal inoculation of sterilehuman disease materials containing Micromyces intracellularis into youngmice systemic illness of varying degrees in the individual mice isproduced. This illness is characterized by subdued spontaneous activity,rough coats, and huddling. Although an occasional mouse dies during thefirst week following intraperitoneal inoculation of the inoculum, mostmice so inoculated recover from this initial phase of postinoculationmorbidity. Two weeks after inoculation, however, increased spontaneousmortality begins to occur in the inoculated mice as compared to controlmice. This increased rate of mortality continues through succeedingweeks of observation at a rate comparable to other routes ofinoculation, i.e., subconjunctival or subcutaneous inoculation. Duringthe ensuing weeks of observation about 5 to 10% of the inoculated micedemonstrate varying degrees of coat changes--alopecia, greasy coats,dull or rough coats, macules or papules--and also demonstrate swollenjoints, locomotor disturbances, seizures, jerky head tremors, or closedeye lids. Sometimes these abnormalities are present for only a few days,but they are usually followed by death of the mouse within a few daysafter their occurrence.

B. Guinea Pigs

During the initial 10 to 12 weeks, following intraperitoneal inoculationof sterile human disease materials containing Micromyces intracellularisinto young guinea pigs produces no observable change in the appearanceof the animal. Thereafter, about 25% of the inoculated guinea pigs beginto demonstrate evidence of systemic illness usually manifested byvarying combinations of the following signs: rapid weight loss, paresis,swollen joints, jerky head movements, and/or marked soft peripherallymphadenopathy. Accelerated mortality, as compared to controls, beginsto occur and usually about 85-100% of the inoculated guinea pigs aredead by the end of the sixth month following inoculation.

The data contained in the following Table II are based on observationusing 200 CF (Carworth Farms) outbred mice, 100 experimental and 100control, for each mode of inoculation. These data provide a generalclassification of the pathologic lesions observed and their incidencewhen the mouse is used for the modelling of chronic human disease due toMicromyces intracellularis.

                  TABLE II                                                        ______________________________________                                                           Incidence                                                  Mode of   General Classification                                                                       Experi- Con-  Chi                                    Inoculation                                                                             of Pathologic Lesions                                                                        mental  trol  Square                                 ______________________________________                                                  Chronic Inflammatory                                                Subconjunctival                                                                         Eye Disease    100%    0%    196.0                                            Degenerative Eye                                                              Disease        75%     0%    116.8                                            Chronic Inflammatory                                                                         25%     0%    26.33                                  Subcutaneous                                                                            Skin Disease                                                                  Degenerative Skin                                                             Disease        25%     0%    26.33                                            Chronic Inflammatory                                                Intraperitoneal                                                                         Systemic Disease                                                                             75%     2%    109.5                                            Degenerative Systemic                                                         Disease        25%     2%    20.72                                  ______________________________________                                    

The date contained in Table III, below, are based on special microscopicpathologic observations in 1000 mice and 100 guinea pigs inoculated intoa variety of sites (e.g., interperitoneal, subcutaneous back andsubconjuctival) with sterile clinical materials containing Micromycesintracellularis. Local inoculation of the agent into each of the organslisted in Table III can be expected to produce a greater incidence ofthe described lesion at the inoculation site.

                                      TABLE III                                   __________________________________________________________________________                              % INCIDENCE                                                                   EXPERIMENTAL                                                                            CONTROL                                                                 GUINEA    GUINEA                                ORGAN    DESCRIPTION OF LESION                                                                          MICE                                                                              PIG   MICE                                                                              PIG                                   __________________________________________________________________________    HEART    Subendocardial Aschoff                                                                         10% 25%   2%  0%                                             Nodules in both mice &                                                        guinea pigs, Subendo-                                                         cardial fibrinoid                                                             necrosis in mice,                                                             sarcomyolysis of                                                              myocardium in guinea                                                          pigs.                                                                SPLEEN   Splenomegaly; acute or                                                                         50% 50%   5%  0%                                             subacute splenitis,                                                           atypical lymphoid                                                             hyperplasis in both mice                                                      and guinea pigs.                                                     SKIN     (SUBCUTANEOUS INCOCULA-                                                                        20%  5%   0%  0%                                             TION)                                                                         Chronic panniculitis,                                                         granulomas, acute and                                                         chronic ulcers, fibrosis,                                                     dermal adnexal atrophy,                                                       necrosis                                                             LYMPH NODES                                                                            Marked reactive lym-                                                                           20% 10%   2%  0%                                             phoid hyperplasia in                                                          mice and guinea pigs,                                                         atypical lymphoid hyper-                                                      plasia in mice, granulo-                                                      matous lymphadenitis in                                                       guinea pigs, necrotic                                                         nodes in guinea pigs.                                                JOINTS   Rheumatoid nodules                                                                             10% 20%   0%  0%                                             around joint cavity in                                                        mice & guinea pigs,                                                           papillary synovitis in                                                        mice, extensive periartic-                                                    ular fibromatosis in guinea                                                   pigs, calcified focal                                                         necrosis in tendons of                                                        guinea pigs, acute and                                                        chronic synovitis in mice                                                     and guinea pigs.                                                     CENTRAL AND                                                                            Focal acute inflamma-                                                                           5%  5%   0%  0%                                    PERIPHERAL                                                                             tion in peripheral                                                   NERVOUS  nerves, cerebral                                                     SYSTEM   cortex, spinal cord                                                           or meninges                                                          EYES     (INTERPERITONEAL                                                              INOCULATION)     20% 20%   0%  0%                                             (SUBCONJUNCTIVAL                                                              INOCULATION)     100%                                                                              50%   0%  0%                                             Acute and chronic                                                             uveal tract inflamma-                                                         tion, optic nerve men-                                                        ingitis, episcleritis,                                                        cataracts, anterior and                                                       posterior synecchia,                                                          interstitial keratitis,                                                       retinopathy, optic                                                            neuritis, orbital pseud-                                                      otumers, extraocular                                                          muscle myositis                                                      __________________________________________________________________________

Based on the above-described details of the animal model and uniquemicrobial agent found in association with the foregoing lesions, theanimal model is useful in the present invention for the development ofdiagnostic materials, such as antigens for in vivo and in vitrodiagnostic tests, and therapeutic agents such as vaccines andchemotherapeutics, for the diagnosis and treatment of disease.

Using the animal model described above, it has been discovered accordingto the present invention that certain chemotherapeutic agents areeffective in protecting the animal model from the significantlyincreased mortality associated with the introduction therein of thepre-tested inoculum. In particular, it has been found thatd-2,2'-(ethylenediimino)-di-1-butanol (also known as "ethambutol") whichhas the formula: ##STR2## 4,4'-sulfonyldianiline (also known as"dapsone") which has the formula: and3-[[(4-methyl-1-piperazinyl)imino]methyl]-rifamycin SV (also known as"rifampin") which has the following formula: ##STR3## are usefulchemotherapeutics in the practice of the present invention, particularlyin the treatment of diseases associated with the above-describedMicromyces intracellularis. These compounds are known compositions ofmatter and procedures for obtaining them will be apparent to thoseskilled in the synthetic organic or pharmaceutical arts.

Thus, ethambutol can be synthesized by heating ethylene dichloride with(+)-2-aminobutanol or, alternatively, by alkylating 2-aminobutanol withglyoxal using NaBH₄ as a reducing agent according to Wilkinson et al.,J. Am. Chem. Soc., 83, 2212 (1961). Pharmaceutical grade ethambutol(hydrochloride) is commercially available from American Cyanamid Co.under the trade name "Myambutol." Likewise, dapsone can be made startingfrom para-chloronitrobenzene according to the method described in U.S.Pat. No. 2,385,899 (1945) and is commercially available in a formsuitable for chemotherapeutic use from Imperial Chemicals Ltd. under thetrade name "Avlosulfon." Finally, rifampin is described in Allen et al.,Drug Intel. Clin. Pharm., 5(11), 364 (1971) and is available in a formsuitable for medicinal use from Giba-Geigy Corp. under the trade name"Rimactane.

DESCRIPTION OF PREFERRED EMBODIMENTS

The following examples are provided for the purpose of furtherillustrating, without limitation, the animal model of the presentinvention and the application of the above-mentioned therapeutic agentsto the treatment of diseases associated with the novel pathogen.

EXAMPLE I

Two hundred male AKR strain mice, ranging in age from 8 to 10 weeks,were obtained from the Jackson Laboratories, Bar Harbor, Maine and heldfor stabilization for a period of two weeks. At the end of this time,the mice were divided equally into five groups, A, B, C, D and E offorty mice each as follows:

Each animal in Groups A, B, C and D was subconjunctivally inoculated ineach eye with a 0.05 milliliter aliquot of pooled, sterile aqueous humorspecimens from patients with active endogenous uveitis and containingMicromyces intracellularis.

Group A mice each thereafter received daily oral dosing with 0.1milliliter of an aqueous suspension containing 25 milligrams ofethambutol per kilogram of body weight for the first two months of theexperiment. For the remaining ten months of the experiment, theconcentration of ethambutol per daily dosage was reduced to 15milligrams per kilogram of body weight.

Group B mice each thereafter received daily oral dosing with 0.1milliliter of an aqueous suspension of 10 milligrams of rifampin perkilogram of body weight, for the duration of the experiment.

Group C mice each thereafter received daily oral dosing with 0.1milliliter of an aqueous solution of 1 milligram of dapsone per kilogramof body weight, for the duration of the experiment.

Group D mice each thereafter received daily oral dosing with 0.1milliliter of sterile, distilled water for the duration of theexperiment.

Each animal in Group E (the "negative control group") wassubconjunctivally inoculated with 0.05 milliliter of sterile isotonicsaline solution. Each mouse in this group thereafter received oraldosing with 0.1 milliliter of sterile, distilled water for the durationof the experiment.

The five groups of animals were observed for 12 months to detectspontaneous mortality. The results at the end of this time were asfollows:

    ______________________________________                                        Group       Absolute Mortality                                                                          % Mortality                                         ______________________________________                                        Group A     24            60                                                  Group B     26            65                                                  Group C     28            70                                                  Group D     34            85                                                  Group E     20            50                                                  ______________________________________                                    

The statistical significance of the mortality rates observed in thisexample is as follows:

Group A versus Group D x² =5.07

Group B versus Group D x² =3.266

Group C versus Group D x² =1.8

Group D versus Group E x² =9.63

Based on these statistical calculations, it can be concluded that thepathogen-inoculated animal model of the present invention exhibits ahighly significant mortality rate compared to saline-inoculated negativecontrol animals. Also, it can be seen that ethambutol, rifampin anddapsone all convey significant protection against the mortality seen inthe animal model.

EXAMPLE II

As an example of the utility of the foregoing three chemotherapeuticagents based on the animal model, it has been found that ethambutol isespecially effective in treating endogenous uveitis. In a noncontrolledstudy over a period of five years, a large number of patients, more than50, were treated with systemic ethambutol. Some patients remainedsymptom free while taking the medicine; when the treatment wasdiscontinued the disease returned in its full severity. Upon restartingthe drug the eye inflammation again resolves in several weeks. Thiscycle was repeatedly observed in many patients over prolonged periods oftime. Other patients have had their long-standing disease cured withprolonged drug treatment.

In a controlled experiment using ethambutol for a double-blindcross-over study, 41 patients were treated and followed for one-year'stime. It was found from a follow-up treatment of 20 patients that thedrug is more effective than placebo in suppressing the disease. Many ofthe patients in this controlled study have had disease of many yearsduration terminated with a few months of therapy.

In addition to the treatment of endogenous uveitis, other diseases canbe treated with the aforementioned chemotherapeutics. Thus, an isotonicaqueous solution of ethambutol instilled daily in the conjunctival sachas been found to be efficacious in relieving signs and symptoms ofchronic conjunctivitis, kerato-conjunctivitis and keratitis. The drughas also been given orally and topically for these conditions andappears to be similarly effective.

The present invention has been described with reference to certainspecific embodiments which have been presented for purposes ofillustration. It is to be understood, however, that numerous variationsof the invention can be made which are well within the scope and spiritof the invention as described in the following claims.

We claim:
 1. A method of treating active endogenous uveitis caused byMicromyces intracellularis comprising administering to a patient havingendogenous uveitis an effective amount therefor of a therapeutic agentcomprising d-2,2'-(ethylenediimino) di-1-butanol.
 2. A method oftreating endogenous uveitis according to claim 1 wherein the therapeuticagent is administered subconjunctivally into the affected eye.
 3. Amethod of treating endogenous uveitis according to claim 1 wherein thetherapeutic agent is administered in the form of an aqueous isotonicsolution.
 4. A method of treating endogenous uveitis according to claim1 wherein the therapeutic agent is administered orally.
 5. A method oftreating endogenous uveitis according to claim 1 wherein the therapeuticagent is administered topically to the affected eye.
 6. A method oftreating chronic conjunctivitis caused by Micromyces intracellulariscomprising administering to a patient having chronic conjunctivitis aneffective amount therefor of a therapeutic agent comprisingd-2,2'-(ethylenediimino)di-1-butanol.
 7. A method of treating chronicconjunctivitis according to claim 6 wherein the therapeutic agent isadministered subconjuctivally into the affected eye.
 8. A method oftreating chronic conjunctivitis according to claim 6 wherein thetherapeutic agent is administered in the form of an aqueous isotonicsolution.
 9. A method of treating chronic conjunctivitis according toclaim 6 wherein the therapeutic agent is administered orally.
 10. Amethod of treating chronic conjunctivitis according to claim 6 whereinthe therapeutic agent is administered topically to the affected eye. 11.A method of treating kerato-conjunctivitis caused by Micromycesintracellularis comprising administering to a patient havingkerato-conjunctivitis an effective amount therefor of a therapeuticagent comprising d-2,2'-(ethylenediimino1(di-1-butanol.
 12. A method oftreating kerato-conjunctivitis according to claim 11 wherein thetherapeutic agent is administered subconjunctivally into the affectedeye.
 13. A method of treating kerato-conjunctivitis according to claim11 wherein the therapeutic agent is administered in the form of anaqueous isotonic solution.
 14. A method of treatingkerato-conjunctivitis according to claim 11 wherein the therapeuticagent is administered orally.
 15. A method of treatingkerato-conjunctivitis according to claim 11 wherein the therapeuticagent is administered topically to the affected eye.
 16. A method oftreating keratitis caused by Micromyces intracellularis comprisingadministering to a patient having keratitis an effective amount thereforof a therapeutic agent comprising d-2,2'-(ethylenediimino)di-1-butanol.17. A method of treating keratitis according to claim 16 wherein thetherapeutic agent is administered subconjunctivally into the affectedeye.
 18. A method of treating keratitis according to claim 16 whereinthe therapeutic agent is administered in the form of an aqueous isotonicsolution.
 19. A method of treating keratitis according to claim 16wherein the therapeutic agent is administered orally.
 20. A method oftreating keratitis according to claim 16 wherein the therapeutic agentis administered topically to the affected eye.